The goal of this project is to integrate an isothermal amplification system with lateral flow detection to produce a simple, sensitive, low cost, all-in-one detection device for flaviviruse that is easily performed and interpreted in a point of care setting and is amenable to use in poorly served communities. The viruses of the family Flaviviridae, e.g. dengue, West Nile, and Japanese Encephalitis, are insect-borne viruses important to both human and veterinary medicine. According to WHO, dengue virus alone affected 1.2 million globally, resulting in 3442 deaths in 1998. These diseases are transmitted by mosquito and ticks and usually are maintained in a transmission cycle in nature. They are widely distributed throughout the world, although a specific flavivirus may be geographically restricted. They produce a broad spectrum of clinical responses in humans ranging from asymptomatic infection to fulminant encephalitis or haemorrhagic fever. Nearly 60 flaviviruses are known to exist, although but many are yet to be shown to cause disease in humans. They present with overlapping clinical signs and symptoms in similar geographic areas. The diagnosis of these infections is currently slow, expensive, and labor intensive and a delay in diagnoses often leads to serious sequelae and sometimes death. Thus, we propose to develop a comprehensive assay specific for several flaviviruses, with initial focus on dengue virus and West Nile virus. These tests are based on isothermal nucleic acid amplification technologies. We will compare the established technology, Loop-mediated AMPlification (LAMP), to our newly developed method SPIDR, which in early studies appears to be easier to implement and affords greater specificity. Both SPIDR and LAMP are isothermal techniques, precluding the need for thermocyclers and can produce results in less than 30 minutes. The amplification products will be processed in an integrated system using lateral flow technology, allowing sensitive, rapid, unambiguous detection with the potential for multiplexing. Once a test is developed, it will be very simple to perform and does not require DNA isolation for sample preparation (as compared to PCR). Such simplified detection facilitates interpretation by less skilled operators. When developed, the device will not require instrumentation or trained scientists to operate and is especially suitable to resource poor settings. The project is proposed by the PI in collaboration with several accomplished scientists across a broad field of synergistic research interests. The proposed research will address a fundamental, program-relevant need for a point of care test useful in developing country settings as well as for rural and socioeconomically disadvantaged Americans. PUBLIC HEALTH RELEVANCE: The goal of this project is to fully integrate a simple, low cost, all-in-one amplification and detection device for flavivirus testing that is easily performed and interpreted in a point of care manner in resource poor settings. The device will not require instrumentation or trained scientists to operate. The development of a reliable, sensitive and specific "point of care" test for flaviviruses in blood samples and other body fluids would be a major breakthrough for many low resource settings.